Joint Application of NMR and Scattering Haydyn Mertens PhD
Methods combining SAXS with NMR Filtering/scoring NMR conformers Direct refinement of structure Refinement of domain/subunit positions
Restraints: SAXS & NMR Orientation Shapes/envelopes RDC s PCS Interface Dimensions (Rg, Dmax) NOEs Chemical Shifts PRE
Restraints: SAXS & NMR Distances: NOEs Chemical Shifts PRE Orientation: RDCs PCS SAXS provides shape/distance info
Model Filtering
Model filtering by SAXS Structures determined X-ray crystallography NMR Homology modeling Docking algorithms Score these models using SAXS data best fit to SAXS curves cluster models
Model filtering by SAXS pyDockSAXS FTDock pyDock SAXS (CRYSOL) ~ 40% improvement in selection of correct solution Pons et al., J Mol Biol. 2010 Oct 22;403(2):217-30
Docking Steps: pyDockSAXS FTDock (http://www.sbg.bio.ic.ac.uk/docking/ftdock.html) generates large pool of complexes Surface complementarity Electrostatics filter pyDOCK (http://www.cllgenome.es/servlet/pydock/) scores/ranks FTDock complexes Binding energy electrostatics desolvation vdw CRYSOL (http://www.embl-hamburg.de/biosaxs/atsas-online/) scores/ranks complexes (fit to SAXS)
Docking Performance: pyDockSAXS Identification of near native solutions 43% 29% 23% 21% Rank (top N predictions for each benchmark member) Pons et al., J Mol Biol. 2010 Oct 22;403(2):217-30 Combined pyDOCK + CRYSOL is best!
Docking Performance: pyDockSAXS Anisometry has an impact on selection Pons et al., J Mol Biol. 2010 Oct 22;403(2):217-30 Spherical complexes hard to select by SAXS
Model filtering by SAXS pyDockSAXS combined scoring function: pyDockSAXS = E pyDOCK + w c X CRYSOL SAXS can aid in the selection of near-native docking models Pons et al., J Mol Biol. 2010 Oct 22;403(2):217-30
Building a model with RDCs & SAXS
Building a model with RDCs & SAXS Basic idea: Use known domain structures Orient domains with RDCs Reduce possible solutions with SAXS domain-distance ( Rg ) scoring/filtering
Example: Calmodulin Calmodulin (CaM) Ca 2+ binding protein (4 EF-hand motifs) Binds multiple targets (multiple functions) Structure is flexible Solved in both extended & compact forms +ligand -ligand Extended form Compact form
Example: Calmodulin-TFP (trifluoperazine) CaM compacts upon TFP binding decreased Dmax observed by SAXS Quaternary structure built from RDCs, domain structures and SAXS SAXS: Rg = 2.0 nm -> Rg = 1.8 nm +TFP Mattinen et al., Biophys J. (2002) 83:1177-1183
Building CaM-TFP model RDCs determine PAS (Azz, Axx, Ayy) D NH from single alignment medium Orientations using free CaM domain structures Mattinen et al., Biophys J. (2002) 83:1177-1183
Building CaM-TFP model 4 degenerate relative orientations SAXS constrains distance between domains ( Rg ) Reduced to a single solution SAXS 1LIN Solutions scored using CRYSOL Mattinen et al., Biophys J. (2002) 83:1177-1183
SASREF can do this too! (in principle)
Using RDCs in SASREF Collect RDCs Define domain orientations PALES (http://www.mpibpc.mpg.de/groups/zweckstetter/_links/software_pales.htm) Xplor-NIH python tools (http://nmr.cit.nih.gov/xplor-nih/) Use pre-oriented PDBs as input rigid bodies Allow only translations in SASREF
Direct Refinement
Direct Refinement Requires accurate and fast calculation of scattering intensity from structure What people currently use: Spherical harmonics (CRYSOL) Zernike potentials (SASTBX) Debye (Xplor-NIH, MODELLER)
Quick reminder: intensity calc Debye: Form factors Distances Need to evaluate all distances Computationally expensive scales quadratically with number of atoms
Quick reminder: intensity calc Improve speed: globbic approximation group atoms (eg. GASBOR)
Quick reminder: intensity calc Multipole expansion (spherical harmonics) Very fast Computation time is linear with size
Quick reminder: intensity calc Direct refinement with NMR & SAXS slow back-calculation of I(s) globbic approx can help reduce number of points bin experimental data less points for calc I(s) Excluded volume treatment Hydration layer treatment
Xplor-NIH (direct refinement)
Xplor-NIH example: E. coli Enzyme-I : HPr complex Active sugar-phosphate transfer in bacteria Phosporylation of E1 and transfer to HPr Significant differences in crystal and NMR structures E1 crystallographic dimers Crystal NMR Schwieters et al., JACS (2010) 132:13026-13045
Xplor-NIH example: Strategy: backbone n-h RDCs show E1 nter subdomain orientations unchanged (free vs complex) thus assuming E1 cter dimerisation domain structure unchanged from E1 crystal structures: orientation E1 dimer from E1 nter RDCs Combine with SAXS/WAXS data Determine free E1 Introduce HPr and determine E1:HPr Schwieters et al., JACS (2010) 132:13026-13045
Xplor-NIH example: Backbone n-h RDCs show E1 nter subdomain orientations unchanged (free vs complex) Schwieters et al., JACS (2010) 132:13026-13045
Xplor-NIH: protocol E1 dimer E1-HPr dimer Schwieters et al., JACS (2010) 132:13026-13045
Comparison to existing structures: Schwieters et al., JACS (2010) 132:13026-13045
Xplor-NIH example: Using SANS to discriminate between 2 possible E1-HPr clusters? 2 H- E1 / 1 H- HPr in 40% D2O (see E1 only) Cluster-1 Cluster-2 Schwieters et al., JACS (2010) 132:13026-13045
Model Mechanism: E1-HPr Schwieters et al., JACS (2010) 132:13026-13045 Relaxation to I through X-ray crystal intermediate (phosphorylated intermediate) Free E1 Relaxation to V through intermediate NMR/SAXS
CNS (direct refinement)
CNS: xafs.f module Direct refinement (RDCs & SAXS) Power series expansion of SAXS curve (FAST) Rg region ---> inter-domain distance Higher angles ---> domain positions Grid-search to account for solvation Gabel et al., JBNMR (2008) 41:199-208
CNS example SAXS + RDCs reduces possible orientations reduces inter-domain translations Gabel et al., JBNMR (2008) 41:199-208
DADIMODO (search algorithm)
DADIMODO Genetic algorithm based Optimise multi-domain structures Adjusts regions of non-defined structure Start from crystal/nmr structure or homology model Objective function (SAXS & RDCs)
DADIMODO Mareuil et al., Eur biophys J (2007) 37:95-104
DADIMODO example gamma-S crystallin vertebrate eye lens component Target Domain Models Conformers (25) Mareuil et al., Eur biophys J (2007) 37:95-104
DADIMODO example gamma-S crystallin Mareuil et al., Eur biophys J (2007) 37:95-104
Some considerations Method of intensity calculation spherical harmonics FAST, accurate (to ~ 5 nm -1 ) Debye SLOW, accurate (to 5-10 nm -1 ) Treatment of solvent Envelope Explicit layer of water Solvent treatment paramount for WAXS
Summary NMR and SAXS/SANS very complementary Scoring Direct refinement Rigid body refinement and docking But if data is rubbish --> model is rubbish! check overall parameters!!!
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